Large changes in the CRAC segment of gp41 of HIV do not destroy fusion activity if the segment interacts with cholesterol.
The membrane-proximal external region (MPER) of the gp41 fusion protein of HIV is highly conserved among isolates of this virus and is considered a target for vaccine development. This region also appears to play a role in membrane fusion as well as localization of the virus to cholesterol-rich domains in membranes. The carboxyl terminus of MPER has the sequence LWYIK and appears to have an important role in cholesterol interactions. We have tested how amino acid substitutions that would affect the conformational flexibility of this segment could alter its interaction with cholesterol. We studied a family of peptides (all peptides as N-acetyl-peptide amides) with P, G, or A substituting for W and I of the LWYIK sequence. The peptide having the greatest effect on cholesterol distribution in membranes was the most flexible one, LGYGK. The corresponding mutation in gp41 resulted in a protein retaining 72% of the fusion activity of the wild-type protein. Two other peptides were synthesized, also containing two Gly residues, GWGIK and LWGIG, and did not have the ability to sequester cholesterol as efficiently as LGYGK did. Making the corresponding mutants of gp41 showed that these other two double Gly substitutions resulted in proteins that were much less fusogenic, although they were equally well expressed at the cell surface. The study demonstrates that drastic changes can be made in the LWYIK segment with the retention of a significant fraction of the fusogenic activity, as long as the mutant proteins interact with cholesterol.
Vishwanathan
Sundaram A
SA
Emory Vaccine Research Center, Yerkes, Emory University, 954 Gatewood Road, Atlanta, Georgia 30329, USA.
Thomas
Annick
A
Brasseur
Robert
R
Epand
Raquel F
RF
Hunter
Eric
E
Epand
Richard M
RM
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002152:Calorimetry, Differential Scanning; D002460:Cell Line; D002522:Chlorocebus aethiops; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism*; D015700:HIV Envelope Protein gp41 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008561:Membrane Fusion; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism
D015700:HIV Envelope Protein gp41; D010446:Peptide Fragments; D002784:Cholesterol